Swietemicrolides A–D, mexicanolide-type limonoids from the bark of Swietenia macrophylla with in vitro cytotoxic and α-glucosidase inhibitory activities

Four new mexicanolide-type limonoids, swietemicrolides A–D (1–4), together with three known compounds (5–7) were isolated from an ethyl acetate extract of the bark of Swietenia microphylla. 1 and 2 had 1,8-hemiacetal systems whilst 3 and 4 shared hexacyclic skeletons consisting of three fused five-membered rings. The structures of the isolated compounds were determined using spectroscopic methods. The five limonoids (1–5) were tested in vitro for their cytotoxic effects against two human cancer cell lines (KB carcinoma and A549 lung cancer cells) and α-glucosidase inhibitory activity. None of them showed significant cytotoxic activity, however, swietemicrolide C (3) exhibited strong effect towards α-glucosidase. Moreover, a possible biosynthetic pathway for compounds 1–4 was proposed to support a comprehensive understanding of the configurations of the new limonoids.


Introduction
Swietenia is a genus belonging to the Meliaceae family, and mainly found in tropical and subtropical Americas.It also grows in China and several Southeast Asian countries such as Vietnam and Indonesia.4][15] Limonoids are iden-tied as major natural products which are characteristic of the genus Swietenia, especially mexicanolide and phragmalin types. 1,15,16 It has not been studied for its phytochemical constituents and biological activities although many investigations on the genus have been performed.In Vietnam, the species is planted to cover bare hills and provide wood for the Southeast of Vietnam.
In the present study, we reported the isolation and structural identication of seven compounds, consisting of four new mexicanolide-type limonoids, named swietemicrolides A-D (1-4), along with a known andirobin-type limonoid characterized as swiemahogin A (5), 19 a steroid, b-sitosterol-3-O-b-D-glucopyranoside (6) 20 and homovanillyl alcohol (7)  21 from the bark (Fig. 1).Compounds 3 and 4 were proved to possess a grandifotane scaffold, a complex hexacyclic skeleton containing three fused ve-membered rings, which has been seen in a unique compound, grandifotane A, so far. 22In addition, the ve limonoids (1-5) were evaluated for their in vitro biological potency comprising cytotoxic activities against KB carcinoma, A549 lung cancer cell lines and a-glucosidase inhibition.

General experimental procedures
Melting points were obtained on a Wagner & Munz Polytherm A hot stage microscope.Optical rotations were measured on an A. Krüss Optronic photometer.UV spectra were recorded with an Agilent 8453 spectrophotometer.IR spectra were determined using a Fisher iS50 FT-IR infrared spectrophotometer.NMR spectra were taken on a Bruker Avance III 500 [500 MHz ( 1 H) and 125 MHz ( 13 C)].HRESIMS was performed on an X500R QTOF spectrophotometer.Column chromatography (CC) was conducted on silica gel (Merck, 40-63 mm, Merck).Thin layer chromatography (TLC) was developed on precoated TLC plates normal phase 60 F254 (Merck, 250 mm) and RP 18 (Merck, 200 mm).TLC plates were visualized using UV lamp or spraying with vanillin-H 2 SO 4 in EtOH and were then heated at about 120 °C for 3-5 min.

Plant material
The bark of Swietenia microphylla was collected in Trang Bom Botanical Garden in January 2019 and identied by Dr Dang Viet Hung, Vietnam National University of Forestry at Dong Nai Province, Vietnam.A voucher sample (SMIB-TB-2019) is deposited in the Natural Product and Medicinal Chemistry Lab, VNUHCM-University of Science.

In vitro cytotoxic assay
The cytotoxic activity of the limonoids were determined using the MTT assay described by Mosmann with ellipticine as the positive control. 23The KB (CCL-17™) and A549 (CCL-185™) cell lines were derived from the American type culture collection (ATCC).The cell lines were kept in liquid nitrogen, activated and maintained in Dulbecco's modied eagle medium (DMEM) containing 7-10% fetal bovine serum (FBS) supplement.The cells were cultured in DMEM at a ratio 1 : 3 in 3-5 days and incubated at 37 °C under 5% CO 2 , 98% humidity for 24 h.The stock solutions were dissolved in DMSO at 2 mg mL −1 .The experiment was conducted with various concentrations of samples (128, 32, 8 and 2 mg mL −1 ) and duplicated for three times.

a-Glucosidase inhibitory assay
The a-glucosidase inhibitory assay was performed according to the method described by Wan et al. with acarbose as the positive control. 24a-Glucosidase, p-nitrophenyl a-D-glycopyranoside (pNPG) and acarbose were purchased from Sigma-Aldrich.A mixture consisting of samples (120 mL, 1 unit mL −1 ), a-glucosidase (20 mL, 1 unit mL −1 ) and phosphate buffer (0.1 M, pH 6.8) were pre-incubated for 15 min at 37 °C in a 96-well plate, followed by addition of p-nitrophenyl a-D-glucopyranoside (5 mM, 20 mL) solution and incubated for further 15 min at 37 °C.The reaction was then stopped by adding 0.2 M Na 2 CO 3 (80 mL) and the emission of amount of p-nitrophenol was determined at 405 nm.The experiment was duplicated for three times.The inhibition was presented by the IC 50 values.1 and 2) and 2D (Fig. 2 and 3) NMR spectroscopic data of 2 closely resembled those of 1, suggesting that 2 was also a mexicanolide-type limonoid with a 1,8-hemiacetal system.The signicant difference was the absence of an acetoxy group in compound 2.This was in agreement with the HRESIMS spectrum which indicated a deprotonated molecule at m/z 619.2385, which was assigned the molecular formula of C 31 H 40 O 13 with one less degree of unsaturation in comparison with that of compound 1.Furthermore, the upeld shi of the oxygenated tertiary carbon (d C 81.0, C-2) in the 13 C NMR spectrum of 2 implied that the acetoxy group observed in compound 1 was replaced by the hydroxy group.The compound was therefore determined as 2 and is named swietemicrolide B.  1 and 2) revealed resonances for a b-substituted furan ring, a methoxy, two acetyls, four tertiary methyls, four methylenes, ve methines, ve carbonyl carbons, six non-hydrogenated sp 3 carbons including two oxygenated tertiary carbons.The spectroscopic features suggested that compound 3 possibly was a limonoid.A comparison of the 1D and 2D NMR of compound 3 with those of grandifotane A 22 indicated that they shared the same skeletal structure.Grandifotane A was isolated from Khaya grandifoliola, which was known as the rst and the unique mexicanolide-type limonoid bearing grandifotane scaffold.Furthermore, to the best of our knowledge, this is the rst time a grandifotane core limonoid has been discovered in the genus Swietenia.The main differences were the presence of the acetoxy group bonded to C-11 (d C 69.0) and the methylene (d H 2.44 and 2.42, d C 32.3, C-6) observed instead of the oxymethine.The methylene was determined by the spin coupling system of H 2 -6 and H-5 in 1 H-1 H COSY spectrum combined with the key HMBC correlations from H 2 -6 to C-5 and C-7 (Fig. 2).The relative conguration of 3 was partially established by the NOESY spectrum (Fig. 3).The NOE correlations of  1 and 2) and 2D (Fig. 2 and 3) NMR spectroscopic data of 4 shared high similarities with those of compound 3 and grandifotane A, 3) correlated with the methyl protons at d H 2.16, to establish the presence of an acetyl group attached to the oxygenated methine carbon C-6.The correlations observed in the 1 H-1 H COSY, HMBC (Fig. 2) and NOESY (Fig. 3) spectra further conrmed the structure and the relative conguration of 4. The structure of compound 4 was therefore characterized to be swietemicrolide D. The possible biosynthetic pathways for 1-4 are proposed in Scheme 1. Fissinolide is likely to be a biogenetic precursor of compounds 1-2, featuring a 1,8-hemiacetal system, and compounds 3-4, which possess a grandifotane scaffold.Hydroxylation at C-11 of ssinolide by a monooxygenase followed by acetylation could result in the formation of iv, which was possibly transformed into v by a dihydroxylation of the D 8,14 double bond to introduce two vicinal hydroxy groups.Compound v could convert into the key intermediate vi via an intramolecular nucleophilic addition between the ketonic carbonyl (C-1) and the hydroxy at C-8 to yield the 1,8-hemiacetal linkage.The C-2 of vi could undergo a hydroxylation to form compound 2, which is then likely acetylated to give compound 1.The formation pathways of compounds 3 and 4 is proposed according to the plausible biosynthesis of grandifotane A. 22 An enzyme-catalyzed Baeyer-Villiger oxidation at the ketonic carbonyl of v would simultaneously result in an ester bridge between C-1 and C-10 in vii, which starts the formation of the unique tricyclodecane ring system.Transesterication followed by protonation of the hydroxy bonded to C-10 and further Wagner-Meerwein rearrangement convert vii via the intermediate viii into compound 3 with the removal of a water molecule to form a bond between C-2 and C-10.Similarly, compound 4 could be possibly formed from ssinolide by undergoing a hydroxylation followed by an acetylation at C-6 to give ix.Hydroxylation at the D 8,14 double bond could introduce a hydroxy at C-8 (x).The grandifotane skeleton is likely designated as in 3 through several steps via the intermediates xi and xii.The biosynthetic pathway proposal could not only explain the formation of the fused ring systems but also support the relative conguration of 1-4 which are consistent with those of previously reported mexicanolides. 22,28he ve limonoids, including swietemicrolides A-D (1-4) and swiemahogin A (5), were evaluated for their in vitro cytotoxicity against two human cancer cell lines, KB epidermal carcinoma and A549 human lung cancer cells using 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay described by Mosmann 23 with ellipticine as the positive  3).Only swietemicrolide C (3) inhibited more than 50% cell growth at the concentration of 128 mg mL −1 and therefore was further tested for the IC 50 values and displayed weak activities (IC 50 = 174.8mM and 189.9 mM, respectively).Moreover, they were accessed for their in vitro a-glucosidase inhibitory effects (Table 3). 24As the result, only swietemicrolide C (3) showed potential activity with an IC 50 value of 253.6 mM, about three times stronger than the positive control, acarbose (IC 50 = 697.7 mM), while the others were found to be completely inactive.The results suggested that the appearance of an acetoxy side chain at C-11 and a hydroxy group at C-14 attached to the grandifotane skeleton increased the effect whilst the presence of an acetoxy group at C-6 could reduce the activity.

Conclusions
In summary, four new members of the B,D-ring seco limonoids named swietemicrolides A-D (1-4) were isolated from the bark of S. microphylla.Compound 3, bearing uncommon three fused ve-membered rings, exhibited strong a-glucosidase inhibitory activity and fairly weak cytotoxicity against KB and A549 human cell lines.The study signicantly contributed to enriching the structural diversity of limonoid derivatives, especially grandifotane class, and provided target subtances for further investigation of biological activities.
Compound 3 was obtained as small white needles, m.p. 152-154 °C, [a] D 25 -56.5 (c 1.1, MeOH).It was assigned the molecular formula of C 31 H 38 O 12 due to its negative peak at m/z 647.2318 [M + HCOO] − in the HRESIMS spectrum, requiring 13 degrees of unsaturation.The UV spectrum showed a maximum at 208 nm and the IR spectrum had characteristic absorption bands at 3502 cm −1 (O-H), 1741 cm −1 (C]O) and 1234 cm −1 (C-O).The 1 H and 13 C NMR spectra of 3 (Tables

Table 1 1
[25][26][27]NMR data for compounds 1-4 in CDCl 3 (d in ppm, J in Hz)[25][26][27]Additionally, the HMBC correlations from a methine proton (d H 5.06, H-11) and methyl protons (d H 1.93) to a carbonyl carbon (d C 169.8) comprehensively pointed to the acetoxy group bonded to the C-11 position.The presence of an acetoxy group assigned at C-3 was determined by cross-peaks between a methine proton at d H 5.52 (d C 75.7, C-3), methyl protons at d H 2.13 (d C 21.1) and a carbonyl carbon at d C 170.6.
25e UV spectrum exhibited a maximum at 208 nm whilst the IR spectrum indicated characteristic absorptions at 3478 cm −1 (O-H), 1742 cm −1 (C]O) and 1222 cm −1 (C-O).The 1 H NMR spectrum of 1 (Table1) displayed the presence of a b-substituted furan ring [d H 7.82 (1H, s), 7.44 (1H, br s) and 6.77 (1H, s)], a methoxy group (d H 3.67, 3H, s), three acetyl moieties (d H 2.13, tence of the hydroxy at C-14 and the acetoxy instead of the hydroxy group attached to C-11 (d C 69.0).The characteristic chemical shis of C-1 at C 108.9 and C-8 at d C 81.4 indicated the presence of a 1,8-hemiacetal motif with an ether bridge between C-8 and C-1.The conguration of the 1,8-hemiacetal and substituents on rings A and B has been described in other related mexicanolides.25-52.1 (c 1.1, MeOH).The UV spectrum showed a maximum at 209 nm and the IR spectrum gave signals at 3446 cm −1 (O-H), 1740 cm −1 (C]O) and 1228 cm −1 (C-O).The 1D (Tables